Malaria in human beings is caused by four species of Plasmodium, P. falciparum, P. vivax, P. ovale and P. malariae. According to a 1986 report of the World Health Organization (WHO). there are almost 100 million cases of malaria infection worldwide. Of these about 1 million, mostly cases of young children who are infected with P. falciparum, are fatal. Because of the appearance of drug resistant parasites and insecticide resistant mosquito vectors, malaria is spreading. Thus, the Indian Health Authorities reported 100,000 cases of malaria in 1962 and 3 million cases, caused mainly by P. vivax, in 1980 (see Bruce-Chwatt, Essential Malariology, 2nd edition, Heinemann, London [1986]).
Recent technical advances have raised hopes that it would soon be possible to produce an antimalarial vaccine which would counteract the growing spread of the disease. Firstly, new methods in the development of malarial vaccines, e.g., the cloning of genes and their expression in microbial host organisms and the use of monoclonal antibodies for antigen identification, can be used. Secondly, long-term cultures of P. falciparum in human red blood cells (Trager et al., Science 193, 673-675 [1976]) have provided a ready source of material for the study of the malaria parasite. More recently, it has become possible to maintain all stages in the life cycle of the parasite in the laboratory (Ponnudurai et al., Trans. R. Soc Trop. Med. Hyg. 76, 812-818 [1982]; Mazier et al., Science 227 440-442 [1985]).
The natural life cycle of P. falciparum has three different stages. In the first stage, mosquitoes introduce sporozoites into the blood vessels of vertebrates during the intake of food. These sporozoites travel via the bloodstream to the liver and invade the hepatocytes of the host. In the second stage, merozoites develop from these sporozoites. These merozoites pass through several multiplication cycles in erythrocytes of the host and then develop to gametocytes. The gametocytes, which are the sexual stage of the parasite, are taken up by mosquitoes when they feed. After fertilization in the stomach of the insect, the gametocytes develop into sporozoites which then travel to the salivary glands of the insect. There, the cycle can begin again.
Sporozoites, merozoites and gametocytes have different antigens. Vaccines can be produced in principle against any of the different stages of the malaria parasite, but it is known that many polypeptides of the parasite are genetically polymorphic, i.e. that the polypeptide changes slightly from generation to generation. This hinders the immunization of vertebrates against malaria using these polypeptides as antigens, since the once-formed antibodies in time can no longer recognize the altered antigens. Accordingly, an ideal vaccine would be one which is directed against a polypeptide of the parasite having an amino acid sequence which is not variable, i.e. against a polypeptide which is genetically stable. It is known that the amino acid sequence (primary structure) of polypeptides which carry out a specific function, such as enzymes, is constant at least in those regions of the primary structure which are important for function.
An example of a genetically stable polypeptide of P. falciparum is the merozoite antigen having the amino acid sequence (I):
MetAsnAlaProLysLysLeuProAlaAsPValAlaGluGluLeuAlaThrThrAla-W-LysLeuValGlnAla GlyLysGlyIleLeuAlaAlaAspGluSerThrGlnThrIleLys LysArgPheAspAsnIleLysLeuGluAsnThrIleGluAsnArgAlaSerTyrArgAsp LeuLeuPheGlyThrLysGlyLeuGlyLysPheIleSerGlyAlaIleLeuPheGluGlu ThrLeuPheGlnLysAsnGluAlaGlyValPro-X-ValAsnLeuLeuHisAsnGluAsn IleIleProGlyIleLysValAspLys-Y-LeuValAsnIleProCysThrAspGluGlu LysSerThrGln-Z-LeuAspGlyLeuAlaGluArgCysLysGluTyrTyrLysAlaGly AlaArgPheAlaLysTrpArgThrValLeuValIleAspThrAlaLysGlyLysProThr AspLeuSerAsnHisGluThrAlaTrpGlyLeuAlaArgTyrAlaSerIleCysGlnGln AsnArgLeuValProIleValGluProGluIleLeuAlaAspGlyProHisSerIleGlu ValCysAlaValValThrGlnLysValLeuSerCysValPheLysAlaLeuGlnGluAsn GlyValLeuLeuGluGlyAlaLeuLeuLysProAsnMetValThrAlaGlyTyrGluCys ThrAlaLysThrThrThrGlnAspValGlyPheLeuThrValArgThrLeuArgArgThr ValProProAlaLeuProGlyValValPheLeuSerGlyGlyGlnSerGluGluGluAla SerValAsnLeuAsnSerIleAsnAlaLeuGlyProHisProTrpAlaLeuThrPheSer TyrGlyArgAlaLeuGlnAlaSerValLeuAsnThrTrpGlnGlyLysLysGluAsnVal AlaLysAlaArgGluValLeuLeuGlnArgAlaGluAlaAsnSerLeuAlaThrTyrGly LysTyrLysGlyGlyAlaGlyGlyGluAsnAlaGlyAlaSerLeuTyrGluLysLysTyr ValTyr PA0 --W-- is Gln or can be absent; PA0 --X-- is Met or Gln; PA0 --Y-- is Gly or Cys and PA0 --Z-- is Gly or Cys.
wherein